Enzyme Pretreatment Shows Opposite Effects on Soil Solidification and Slurry Dewatering

Excavation, tunnelling, dredging, and underground construction generate enormous volumes of fine-grained soil and high-water-content slurry that must be dewatered, stabilized, or both before transport, reuse, and disposal. When the excavated material is rich in humic substances, the central difficulty is not simply low intrinsic strength or excess water, but persistent organic interference at reactive interfaces. Carboxyl, phenolic, and other oxygen-containing groups in humic matter adsorb on mineral and binder surfaces, complex calcium- and aluminum-bearing species, retain water, and alter particle associations. In compacted binder-treated soils, these effects restrict particle-binder contact, suppress hydration-product development, and weaken the continuity of the load-bearing cemented network. Increasing cementitious reagent dosage can partly compensate for this inhibition, but it does not remove the interfacial cause of the problem, which is why researchers have been searching for ways to modify the humic-rich interface before binder addition or coagulation rather than relying solely on higher reagent dosages.

A new study published in Frontiers in Bioengineering and Biotechnology by Xuanyu Chen, Prithvendra Singh, Kaiwei Luo, Qianwei Ma, and Ningjun Jiang of Southeast University’s Institute of Geotechnical Engineering, working with the Jiangsu Key Laboratory of Low Carbon and Sustainable Geotechnical Engineering and the Indian Institute of Technology Bombay, has now demonstrated that laccase-based enzymatic pretreatment produces strikingly different engineering responses depending on whether the humic-rich soil is being solidified or dewatered. The research team used an artificial humic-rich soil containing 10 percent humic acid, classified as organic clay under the Unified Soil Classification System, to compare laccase pretreatment in composite-binder solidification and slurry dewatering. The artificial soil was prepared from 18 percent quartz-rich silt, 27 percent kaolin, 45 percent calcium bentonite, and 10 percent commercial humic acid, providing a reproducible humic-rich model system for comparing the tested treatment routes.

Laccase is well suited to modifying organic interfaces because it oxidizes a broad range of phenolic and aromatic substrates under mild aqueous conditions using molecular oxygen as the terminal electron acceptor. Its effective reaction domain can be extended with a low-molecular-mass redox mediator such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), commonly abbreviated ABTS, which transfers oxidative equivalents from the enzyme to less accessible substrates. For humic-rich soil, however, a larger reaction domain cannot be assumed to be universally beneficial. Humic substances themselves participate in laccase-driven transformations and can also aggregate with or inhibit laccase, so enzyme performance depends on the physicochemical environment in which the catalytic system operates. The engineering question is therefore not whether ABTS increases laccase reactivity in isolation, but whether the resulting spatial pattern of organic transformation is favorable for the subsequent engineering process.

The experimental program comprised four parts: a laccase-dose comparison for solidification, a component-comparison test of laccase alone, laccase plus ABTS, and laccase plus ABTS plus carboxymethyl chitosan, bench-scale coagulation and flocculation with vacuum filtration, and geotextile-tube dewatering. For solidification, the treated soil was mixed with 8 percent of a self-developed calcium-rich composite curing agent consisting of carbide residue, gypsum, and Portland cement at a dry-mass ratio of 20:1:1 after 24 hours of pretreatment. For dewatering, the same solids were dispersed as a high-water-content slurry with an initial water content of 383.9 percent on a dry-mass basis and conditioned with polyaluminum chloride followed by carboxymethyl chitosan or cationic polyacrylamide.

The solidification results were unambiguous. At laccase dosages of 20,000 and 40,000 units per kilogram of dry soil, the 14-day unconfined compressive strength reached 841.4 and 818.3 kilopascals respectively, compared with only 434.9 kilopascals for the untreated control. The 20,000-unit dosage was therefore selected for subsequent experiments because increasing the nominal laccase input to 40,000 units per kilogram did not provide additional strength improvement. In the component-comparison test, the 14-day strength was 797.4 kilopascals with laccase alone, 630.6 kilopascals with laccase plus ABTS, 518.0 kilopascals with laccase plus ABTS plus carboxymethyl chitosan, and 410.6 kilopascals for the control. Laccase alone combined the highest 14-day strength with the lowest axial expansion of 1.74 percent, compared with 2.58 percent for the control. The addition of ABTS, and particularly the subsequent incorporation of carboxymethyl chitosan, progressively diminished the strengthening effect rather than providing an additive benefit.

Microstructural evidence from the solidification branch supported this interpretation. Scanning electron microscopy showed that the 14-day laccase-treated specimen had the densest interparticle morphology, with particle surfaces more extensively covered by cementitious products and adjacent particles connected by more continuous solid bridges. Energy-dispersive X-ray spectroscopy revealed that the calcium content increased from 9.63 weight percent in the control to 13.65 weight percent in the laccase-treated specimen, with the atomic calcium-to-silicon ratio rising from 0.410 to 0.774. X-ray diffraction patterns showed broadly similar crystalline-phase assemblages dominated by quartz, calcite, and portlandite, indicating that enzymatic pretreatment did not alter the principal aluminosilicate mineral framework but instead produced treatment-dependent differences in the relative prominence of existing calcium-bearing reflections.

In contrast, mediator addition benefited slurry treatment. In the bench-scale tests, laccase plus ABTS followed by polyaluminum chloride and carboxymethyl chitosan reduced supernatant turbidity to 3.73 nephelometric turbidity units, the lowest value among the chitosan-based formulations, and reduced vacuum-filtrate turbidity to 81.7 units while decreasing cake water content to 57.66 percent. Notably, two formulations with similar zeta potentials of approximately -7 millivolts showed markedly different turbidity and cake water content, indicating that charge reduction alone did not determine clarification performance. Once electrostatic repulsion is reduced, aggregate architecture, floc strength, compressibility, and pore connectivity become increasingly important in controlling dewaterability.

The difference between formulations became even more pronounced during geotextile-tube drainage. Although polyaluminum chloride plus cationic polyacrylamide produced the fastest initial release of water, releasing 725 milliliters within the first 5 minutes, the laccase plus ABTS plus polyaluminum chloride plus carboxymethyl chitosan route subsequently achieved the greatest cumulative water release. At 30 hours, the cumulative filtrate volume reached 1,702.8 milliliters for the enzymatic route, compared with 1,340 milliliters for polyaluminum chloride plus carboxymethyl chitosan and 1,392 milliliters for polyaluminum chloride plus cationic polyacrylamide, representing increases of 27.1 percent and 22.3 percent respectively. The final central-region cake water content was 84.94 percent for the enzymatic route, substantially lower than 112.19 percent for the chitosan route and 127.23 percent for the polyacrylamide route. The directly collected filtrate from the enzymatic route also remained relatively clear at 9.99 turbidity units, compared with 82.86 units for the chitosan route without biochemical pretreatment.

Dissolved organic carbon analysis revealed a paradoxical finding. The DOC concentration decreased from 40.50 milligrams of carbon per liter in untreated soil to 29.07 after laccase treatment, a reduction of 28.2 percent, and further decreased to 20.33 milligrams of carbon per liter after ABTS addition, representing a reduction of 49.8 percent relative to untreated soil. Yet the corresponding 14-day strength decreased from 797.4 to 630.6 kilopascals. Thus, maximizing the reduction in extractable dissolved organic carbon was not equivalent to maximizing solidification performance. The authors suggest that differences in the location or form of organic-matter transformation influenced subsequent particle-binder interactions, with laccase alone producing a more favorable interfacial condition for cementation while ABTS-assisted treatment extended organic transformation over a broader fraction of the system.

The contrasting effects of ABTS on solidification and dewatering reflect the different structural requirements of the two engineering processes. In composite-binder solidification, performance depends on the development of mechanically effective particle-binder contacts during curing, whereas slurry dewatering depends on aggregate formation and the persistence of water-conducting pathways during filtration and cake consolidation. In compacted soil, strength develops primarily at particle-binder contacts; in slurry, organic matter and mineral particles are dispersed throughout the aqueous phase, providing conditions for ABTS-mediated electron transfer to extend organic modification beyond the immediate enzyme-accessible region. ABTS therefore had opposite engineering effects in the two processes: it reduced solidification performance relative to laccase alone but enhanced prolonged chitosan-based slurry dewatering. The authors conclude that the enzyme-mediator combination should be selected according to the intended engineering process rather than treated as universally additive, and they note that validation with natural humic-rich soils and sediments will further define the range of applicability of these formulation trends.

Subject of Research: Enzymatic soil pretreatment with laccase and ABTS shows opposite effects on solidification and dewatering of humic-rich soil

Article Title: Differential effects of ABTS-assisted laccase pretreatment on solidification and dewatering of humic-rich soil

Article References: Chen, X., Singh, P., Luo, K., Ma, Q., & Jiang, N. (2026). Differential effects of ABTS-assisted laccase pretreatment on solidification and dewatering of humic-rich soil. Frontiers in Bioengineering and Biotechnology, 14, Article 1996007. https://doi.org/10.3389/fbioe.2026.1996007

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1996007

Keywords: laccase, ABTS, humic-rich soil, soil solidification, slurry dewatering, redox mediator, geotextile tube, unconfined compressive strength, calcium carbide residue, dissolved organic carbon, enzymatic pretreatment, biogeotechnics